The goal of this proposal is to elucidate molecular mechanisms by which H1 linker histone regulates chromatin structure and activity. H1 and the four core histones (H2A, H2B, H3 and H4) are essential structural proteins of metazoan chromosomes. Much of our knowledge about H1's roles in chromatin is derived from in vitro studies, whereas our understanding of its functions in vivo remains very incomplete. We propose to use the fruit fly, Drosophila melanogaster, as a model system because it provides many advantages for studies of chromosome structure and genetic activity and because Drosophila H1 strongly resembles mammalian H1 linker histones. We developed an RNA interference (RNAi) approach to deplete Drosophila H1 nearly completely in vivo. Using this approach, we showed that H1 is essential for Drosophila development and for normal chromosome architecture. We also discovered that, in addition to its important structural role in chromatin, H1 plays an active role in regulating epigenetic marking and silencing of heterochromatin by directly interacting with and helping to recruit a core histone - modifying enzyme to chromatin. We also found that H1 has a role in controlling DNA replication of polytene chromsomes. To gain a deeper understanding of the molecular mechanisms by which H1 controls nuclear functions, we propose to: (1) Characterize the functional role of H1 in regulating DNA replication; (2) Understand how H1 interfaces with core histone posttranslational modifications in chromatin; (3) Determine the contributions of H1 structural domains and post-translational modifications to its biological functions. The successful completion of this project will help define the mechanisms by which this major constituent of chromosomes partners with other cellular factors to regulate the structure and activity of chromatin.